Breast cancer carboplatin drug-resistant cell strain and construction method thereof

Abstract

The invention relates to the technical field of cell engineering, in particular to a breast cancer carboplatin drug-resistant cell strain and a construction method thereof, the breast cancer carboplatin drug-resistant cell strain is preserved in Guangdong Microbial Culture Collection Center on October 16, 2024, the preservation number is GDMCC No: 6528, and the preservation number is CGMCC NO: 6528. The drug-resistant cell strain MDA-MB-231 / CBP constructed in the invention can stably grow and pass in a carboplatin culture system with an action concentration of 20 [mu] M, has a carboplatin resistance index (RI) of 10.7, provides a basis for research of a carboplatin drug-resistant mechanism and development of related targets, and has a wide application prospect. The invention provides a drug-resistant cell model for researching the drug resistance mechanism of human breast cancer cells to carboplatin, searching an effective treatment method for overcoming human breast cancer drug resistance, preparing antitumor drugs, screening tumor drug resistance reversal drugs, guiding clinical medication and the like, and has important effects and good application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of cell engineering, and in particular to a breast cancer carboplatin-resistant cell line and a construction method thereof. Background Art

[0002] The mammary gland is part of the female reproductive system and is located in the chest. It is responsible for the secretion of milk. Breast cancer is a malignant tumor that originates in breast tissue and usually develops slowly. It may form a small non-malignant mass at the beginning, called a benign breast lesion, and gradually evolve into cancer.

[0003] Carboplatin is a platinum chemotherapy drug that can be used to treat breast cancer. Its chemical formula is: 6 H 12 N 2 O 4 Pt, its structural formula is as follows:

[0004]

[0005] Carboplatin binds to DNA, interferes with DNA replication and repair, and causes cancer cell death. However, carboplatin resistance limits its application in the clinical treatment of breast cancer, and the problem of carboplatin resistance deserves further attention. The occurrence of carboplatin resistance means that the tumor has weakened or completely lost its response to the drug. At present, the specific mechanism of carboplatin resistance is not fully understood, but there are some common mechanisms and influencing factors: (1) Cell signaling pathway variation: Abnormal signaling pathways within breast cancer cells may lead to reduced therapeutic effects of carboplatin, including TP53 mutations, BRCA mutations, and other genetic abnormalities related to cell proliferation and survival. (2) Drug metabolism and excretion: Individual differences in patients may play a role in drug metabolism and excretion, affecting the concentration and duration of drugs in the body, which may lead to reduced efficacy of carboplatin or the development of resistance. (3) Tumor microenvironment: Various cells and molecules in the tumor microenvironment can interact with drugs, thereby affecting the efficacy of drugs, for example, reducing the concentration of drugs in the tumor, providing protective pathways, or promoting molecular signals of resistance.

[0006] Therefore, although the existing technology has revealed the potential of inhibiting some metabolic pathways to overcome carboplatin resistance, the current treatment methods still have the defect of unclear resistance mechanism. Therefore, it is necessary to further study and improve the treatment strategy based on carboplatin-resistant cell lines of breast cancer, such as multi-target combined treatment, solving the inconsistency of treatment response, etc., to improve the effectiveness and tolerance of carboplatin treatment and bring better clinical efficacy to patients. However, there are currently no carboplatin-resistant breast cancer cell lines, which cannot meet the further research of carboplatin resistance in breast cancer. Summary of the invention

[0007] The purpose of the present invention is to provide a breast cancer carboplatin-resistant cell line and a construction method thereof in response to the above-mentioned problems. The resistant cell line is specially developed for the carboplatin resistance of human breast cancer cells, provides an important experimental tool for the study of carboplatin resistance, helps to deeply understand the carboplatin resistance mechanism, and provides a basis for the development of more effective breast cancer treatment strategies.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a breast cancer carboplatin-resistant cell line, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 16, 2024, with a collection number of GDMCC No: 65284.

[0009] The present invention also provides a method for constructing a carboplatin-resistant breast cancer cell line, which comprises: inducing human breast cancer cells to grow and propagate in a carboplatin environment ranging from low concentration to high concentration by combining a method of gradually increasing drug concentration, a high-dose shock method and a high-concentration serum recovery method; in the process of increasing the carboplatin concentration, intermittently using a high-dose carboplatin shock treatment on the cells, and restoring the cell state in a high-concentration serum culture medium; and repeating the induction cycle to finally obtain the drug-resistant cell line.

[0010] Stepwise Increasing Dose is a commonly used method in drug therapy to increase patients' tolerance and adaptability to drugs. The basic principle is to gradually increase the drug dose to allow the patient's body to adapt to the effects of the drug and reduce the occurrence of adverse reactions. This method is usually suitable for starting new drugs or adjusting the dose of existing drugs, especially those with potential toxicity or requiring an adaptation period.

[0011] The Stress Selection Method refers to the process of gradually increasing the drug concentration and intermittently treating the cells with a higher dose of carboplatin (for example, a concentration higher than IC50) for a short time, applying a strong drug shock and rapidly increasing the drug dose to a higher level. This large-dose drug shock can kill most sensitive cells in a short period of time, while the few remaining surviving cells often have stronger drug resistance potential.

[0012] Recovery in High Serum Concentration refers to increasing the serum concentration in the culture medium from the usual 10% to 20% after the rapid increase in the dose shock method in order to help the surviving cells recover quickly and enhance their drug resistance. This high-concentration serum environment can provide rich nutritional support for cells, promote rapid cell growth and metabolism, and also increase the survival rate of cells under high drug pressure.

[0013] Preferably, the construction method specifically comprises the following steps:

[0014] (1) Initial culture: Human breast cancer cells were inoculated in a culture medium containing 10% fetal bovine serum and incubated at 37°C and 5% CO 2 Initial culture is carried out in a saturated humidity incubator. After a period of culture, when the cells are in good condition and adapted to the culture environment, the initial culture solution is obtained;

[0015] (2) Phase I Carboplatin Induction: Carboplatin was added to the initial culture medium at an initial concentration of 0.5 μM. As the cells grew, the concentration of carboplatin was gradually increased by 0.3 μM each time until the concentration reached 2 μM. During this process, the proliferation and survival of cells needed to be continuously monitored to ensure that the cells could grow and propagate normally under the gradually increasing drug pressure.

[0016] (3) First stage shock: the concentration of carboplatin was instantly increased to 20 μM, and the cells were treated at this concentration for 24 hours; the culture medium was then replaced with a culture medium containing 20% ​​fetal bovine serum, and the cells were allowed to recover in a drug-free environment for 72 hours; the culture medium was then replaced with a culture medium containing 2 μM carboplatin, and the cells were cultured again to ensure that the cells retained their drug resistance during the recovery process after the shock;

[0017] (4) Second stage: Carboplatin induction: Cells are cultured stably in a medium containing 2 μM carboplatin for one week, and then the carboplatin concentration is gradually increased by 1 μM each time until the carboplatin concentration reaches 10 μM. During this process, the drug resistance of cells will gradually increase, and the culture medium needs to be replaced regularly to maintain a stable drug concentration.

[0018] (5) Second stage shock: The concentration of carboplatin was instantly increased to 100 μM, and the cells were treated at this concentration for 24 hours. The culture medium was then replaced with a medium containing 20% ​​fetal bovine serum, and the cells were allowed to recover in a drug-free environment for 72 hours. The culture medium was then replaced with a medium containing 10 μM carboplatin and the cells were cultured continuously.

[0019] (6) The third stage of carboplatin induction: After the cells have been cultured in a 10 μM carboplatin environment for one week, the concentration is increased by 2 μM each time until the carboplatin concentration reaches 20 μM, ensuring that the cells can grow and propagate normally in the 20 μM carboplatin culture medium to obtain the target cell line; at this point, the target cell line is obtained, and its drug resistance is significantly improved, and it can survive in a high-dose carboplatin environment;

[0020] (7) Obtaining a stable cell line: The target cell line was repeatedly cultured in a 20 μM carboplatin environment, followed by two freezing and thawing treatments, and finally a stable carboplatin-resistant cell line was obtained.

[0021] Preferably, the specific operation of the cryopreservation and resuscitation treatment in step (7) is: cryopreserving the target cell line in liquid nitrogen for two weeks, then resuscitating and continuing to culture in a culture medium containing 20 μM carboplatin for one week.

[0022] The main functions of step (7) are as follows:

[0023] ① Stability verification: Through repeated freezing and re-cultivation, it can be verified whether the resistant cell line can maintain stable drug resistance after freezing and thawing. This is very important because the cells may have a certain stress response during freezing and the drug resistance may change. By repeating the freezing and re-cultivation process, the stability of drug resistance can be ensured.

[0024] ②Study on drug resistance mechanism: By repeating the process of freezing and re-cultivation, cell samples at multiple time points can be obtained. In this way, cell samples at different time points can be compared to further study the mechanism of cell resistance to drugs. Comparison of cells at different time points may reveal some changes or mutations, which will help reveal the details of the drug resistance mechanism.

[0025] ③Cell preservation: Cryopreservation of cell samples is a common method for preserving cell lines. Regular cryopreservation can ensure the long-term storage and use of drug-resistant cell lines, which is very important for future experimental design, further research, and sharing of cell lines with other laboratories.

[0026] By repeating the freezing and re-cultivation process, the drug resistance of the cell line can be verified and consolidated, and stable drug-resistant cell lines can be provided for subsequent functional and mechanism studies.

[0027] In addition, the present invention also proposes an application of a breast cancer carboplatin-resistant cell line, which are respectively an application of a breast cancer carboplatin-resistant cell line in preparing a resistant cell model, an application of a breast cancer carboplatin-resistant cell line in screening and preparing anti-breast cancer drugs, and an application of a breast cancer carboplatin-resistant cell line in drug target development.

[0028] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0029] 1. The drug-resistant cell line MDA-MB-231 / CBP constructed by the present invention can stably grow and propagate in a culture system with an action concentration of 20 μM carboplatin, and has a resistance index (RI) to carboplatin of 10.7, which provides a basis for the study of carboplatin resistance mechanism and the development of related targets, and provides a drug-resistant cell model for studying the resistance mechanism of human breast cancer cells to carboplatin, finding effective treatment methods to overcome human breast cancer resistance, preparing anti-tumor drugs, screening tumor resistance reversal drugs and guiding clinical drug use, etc., which has an important role and good application prospects.

[0030] 2. In the process of constructing the drug-resistant cell line MDA-MB-231 / CBP, the present invention uses a combination of gradually increasing drug concentration, rapidly increasing dose shock and high-concentration serum recovery methods, and the construction method is simple and easy. Moreover, through the combined effects of gradually increasing drug concentration and high-dose shock, cells can be promoted to develop stronger drug resistance; the rapidly increasing dose shock method can quickly eliminate sensitive cells, and the high-concentration serum recovery method helps the rapid recovery and proliferation of drug-resistant cells; high-concentration serum provides additional nutritional support for drug-resistant cells and reduces cell death after drug shock; this combined method is closer to the actual mode of drug use in clinical treatment, and is helpful to study the development of drug resistance in actual treatment; through repeated cycles of induction, the stability of drug-resistant cell lines and the repeatability of experimental results can be ensured. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The STR identification chart of the drug-resistant cell line MDA-MB-231 / CBP was constructed in Example 1 of the present invention.

[0032] Figure 2 The cell morphology of the drug-resistant cell line MDA-MB-231 / CBP constructed in Example 1 of the present invention under an inverted phase contrast microscope.

[0033] Figure 3 The figure is a growth curve diagram of human breast cancer cell MDA-MB-231 and the drug-resistant cell line MDA-MB-231 / CBP constructed in Example 1 of the present invention.

[0034] Figure 4 The drug concentration-cell inhibition rate curve of the human breast cancer cell MDA-MB-231 of the present invention and the drug-resistant cell line MDA-MB-231 / CBP constructed in Example 1 of the present invention. DETAILED DESCRIPTION

[0035] In order to express the present invention more clearly, the present invention is further described below through specific examples.

[0036] Example 1: Construction of a carboplatin-resistant breast cancer cell line

[0037] The method is constructed by combining a method of gradually increasing drug concentration, a high-dose shock method and a high-concentration serum recovery method, that is, human breast cancer cells are induced to grow and passage in an environment of carboplatin from low concentration to high concentration. During the process of increasing the carboplatin concentration, the cells are intermittently treated with a high-dose carboplatin shock, and the cells are restored to a high-concentration serum culture medium. The induction cycle is repeated to finally obtain the drug-resistant cell line.

[0038] The specific construction method includes the following steps:

[0039] (1) Initial culture: Human breast cancer cells were inoculated in a culture medium containing 10% fetal bovine serum and incubated at 37°C and 5% CO 2 Initial culture is carried out in a saturated humidity incubator. After a period of culture, when the cells are in good condition and adapted to the culture environment, the initial culture solution is obtained;

[0040] (2) Phase I Carboplatin Induction: Carboplatin was added to the initial culture medium at an initial concentration of 0.5 μM. As the cells grew, the concentration of carboplatin was gradually increased by 0.3 μM each time until the concentration reached 2 μM. During this process, the proliferation and survival of cells needed to be continuously monitored to ensure that the cells could grow and propagate normally under the gradually increasing drug pressure.

[0041] (3) First stage shock: the concentration of carboplatin was instantly increased to 20 μM, and the cells were treated at this concentration for 24 hours; the culture medium was then replaced with a culture medium containing 20% ​​fetal bovine serum, and the cells were allowed to recover in a drug-free environment for 72 hours; the culture medium was then replaced with a culture medium containing 2 μM carboplatin, and the cells were cultured again to ensure that the cells retained their drug resistance during the recovery process after the shock;

[0042] (4) Second stage: Carboplatin induction: Cells are cultured stably in a medium containing 2 μM carboplatin for one week, and then the carboplatin concentration is gradually increased by 1 μM each time until the carboplatin concentration reaches 10 μM. During this process, the drug resistance of cells will gradually increase, and the culture medium needs to be replaced regularly to maintain a stable drug concentration.

[0043] (5) Second stage shock: The concentration of carboplatin was instantly increased to 100 μM, and the cells were treated at this concentration for 24 hours. The culture medium was then replaced with a medium containing 20% ​​fetal bovine serum, and the cells were allowed to recover in a drug-free environment for 72 hours. The culture medium was then replaced with a medium containing 10 μM carboplatin and the cells were cultured continuously.

[0044] (6) The third stage of carboplatin induction: After the cells have been cultured in a 10 μM carboplatin environment for one week, the concentration is increased by 2 μM each time until the carboplatin concentration reaches 20 μM, ensuring that the cells can grow and propagate normally in the 20 μM carboplatin culture medium to obtain the target cell line; at this point, the target cell line is obtained, and its drug resistance is significantly improved, and it can survive in a high-dose carboplatin environment;

[0045] (7) Obtaining a stable cell line: The target cell line was repeatedly cultured in a 20 μM carboplatin environment, followed by two freezing and resuscitation treatments. The specific operations of the freezing and resuscitation treatments were as follows: the target cell line was frozen in liquid nitrogen for two weeks, then revived and continued to be cultured in a culture medium containing 20 μM carboplatin for one week to ensure stable drug resistance. Finally, a stable carboplatin-resistant cell line was obtained, named MDA-MB-231 / CBP. The cell line was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, with the deposit number GDMCC No: 65284.

[0046] The MDA-MB-231 / CBP cell line constructed in this example was subjected to STR identification. Figure 1 As shown, Figure 1 The results showed that no site had more than two allele peaks among the 21 sites detected, indicating that MDA-MB-231 / CBP belongs to the MDA-MB-231 cell line.

[0047] Example 2: Morphological observation of carboplatin-resistant breast cancer cell line MDA-MB-231 / CBP

[0048] In this example, the morphological characteristics of the drug-resistant cell line MDA-MB-231 / CBP prepared in Example 1 were observed.

[0049] 1. Experimental methods:

[0050] The morphology of MDA-MB-231 before and after carboplatin induction was observed using an inverted phase contrast microscope and photographed and recorded.

[0051] The groups are as follows:

[0052] (A) is MDA-MB-231, a human breast cancer cell line before induction;

[0053] (B) is MDA-MB-231 / CBP, a drug-resistant cell line obtained after induction with carboplatin.

[0054] 2. Experimental results:

[0055] (1) Figure 2 As shown in Figure A, MDA-MB-231 cells are relatively long spindles with irregular shapes, such as polygonal or spindle-shaped.

[0056] (2) Figure 2 As shown in Figure B, MDA-MB-231 / CBP showed island growth, which was different from the scattered growth before drug resistance. Its morphology also changed to a certain extent. The cell morphology was more disordered, the edges were more irregular, and there were more pseudopodia or protrusions, indicating that after carboplatin-induced resistance, the morphology of the resistant cell line changed significantly.

[0057] Embodiment 3:

[0058] In this example, the growth curve of the drug-resistant cell line MDA-MB-231 / CBP prepared in Example 1 was determined.

[0059] 1. Experimental methods:

[0060] (1) MDA-MB-231 and its drug-resistant cells MDA-MB-231 / CBP in the logarithmic growth phase were digested with trypsin and then resuspended in DMEM complete culture medium;

[0061] (2) The cell density was adjusted to 2 × 104 cells / ml and inoculated in a 96-well plate (100 μL / well). Five replicate wells were set up for each group and incubated at 37°C and 5% CO. 2 After 24 hours, 10 μl CCK-8 solution was added to each well and the cells were placed in a cell culture incubator for 30 min. For the other groups, 10 μl CCK-8 solution was added to each well after 24 h, 48 h, 72 h, 96 h, and 120 h. The absorbance at 450 nm was measured and the cell growth curve was drawn. The results are shown in the table. Figure 3 .

[0062] 2. Experimental results:

[0063] like Figure 3 As shown in the figure, within 2 days of culture, there was no significant difference between the proliferation curve of MDA-MB-231 / CBP cells and that of MDA-MB-231 cells; after 3 days of culture, the proliferation curves of MDA-MB-231 / CBP cells and those of MDA-MB-231 cells were significantly different, with the proliferation rate of MDA-MB-231 cells increasing, while that of MDA-MB-231 / CBP cells was relatively slow. MDA-MB-231 / CBP cells have good proliferation ability.

[0064] Embodiment 4:

[0065] In this example, the drug concentration-survival rate curve was drawn and the drug resistance index was determined for the drug-resistant cell line MDA-MB-231 / CBP prepared in Example 1.

[0066] 1. Experimental methods:

[0067] (1) MDA-MB-231 and MDA-MB-231 / CBP cells in the logarithmic growth phase were taken, the cell density was adjusted to 2×104 cells / ml, and inoculated into a 96-well culture plate;

[0068] (2) Add appropriate amount of carboplatin to make the final concentrations of 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM and 640 μM, respectively. Set up three replicate wells for each concentration and place the wells at 37°C and 5% CO. 2 After culturing in the incubator for 72 h, the absorbance OD of each group at 450 nm was determined by the CCK8 method, and the survival rate (%) was calculated according to the following formula:

[0069] Cell survival rate (%) = sample OD mean / control OD mean × 100;

[0070] The drug concentration-survival rate curve was drawn with the concentration of carboplatin as the horizontal axis and the survival rate of each group as the vertical axis, and the half-inhibitory concentration (IC50) was calculated. The drug resistance index (RI) was calculated according to the following formula.

[0071] Resistance index (RI) = IC50 of resistant cells / IC50 of parental cells.

[0072] 2. Experimental results:

[0073] The experimental results are as follows Figure 4 As shown, Figure 4 Figure A is the drug concentration-cell inhibition rate curve of MDA-MB-231 / CBP. Figure 4 As can be seen in Figure A, the survival rate of MDA-MB-231 / CBP cells in the presence of the same concentration of carboplatin was significantly higher than that of MDA-MB-231 cells;

[0074] Figure 4 Figure B is a drug concentration-cell inhibition rate curve of human breast cancer cell MDA-MB-231. Figure 4 As can be seen from Figure B, MDA-MB-231 cells are more sensitive to low-dose carboplatin, and their survival rate gradually decreases as the concentration increases.

[0075] By calculation, the IC50 of MDA-MB-231 cells is 48.11 μM, and the IC50 of MDA-MB-231 / CBP cells is 514.8 μM. Moreover, the resistance index (RI) of the drug-resistant cell line MDA-MB-231 / CBP constructed by the present invention to carboplatin is 10.7, indicating that the drug-resistant cells have good tolerance to carboplatin, indicating that the MDA-MB-231 / CBP resistant cell line was successfully constructed.

[0076] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A breast cancer carboplatin-resistant cell line, characterized in that: The breast cancer carboplatin-resistant cell line was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 16, 2024, with the collection number GDMCC No: 65284.

2. The method for constructing a carboplatin-resistant breast cancer cell line according to claim 1, characterized in that: The method of gradually increasing drug concentration, the method of rapidly increasing drug dose shock and the method of high-concentration serum recovery are used in combination to induce human breast cancer cells to grow and passage in an environment of carboplatin from low concentration to high concentration. During the process of increasing the carboplatin concentration, the cells are intermittently treated with high-dose carboplatin shock and restored to the cell state in a high-concentration serum culture medium. The induction cycle is repeated to finally obtain the drug-resistant cell line.

3. The construction method according to claim 2, characterized in that: The specific steps include: (1) Initial culture: human breast cancer cells were inoculated into a culture medium containing 10% fetal bovine serum and initially cultured in an incubator at 37° C. and 5% CO 2 saturated humidity to obtain an initial culture medium; (2) First stage carboplatin induction: Carboplatin was added to the initial culture medium, and the initial concentration of carboplatin was 0.5 μM. As the cells grew, the concentration of carboplatin was gradually increased by 0.3 μM each time until the concentration reached 2 μM. (3) First-stage shock: The concentration of carboplatin was instantly increased to 20 μM, and the cells were treated at this concentration for 24 hours; the culture medium was then replaced with a culture medium containing 20% ​​fetal bovine serum, and the cells were allowed to recover in a drug-free environment for 72 hours; the culture medium was then replaced with a culture medium containing 2 μM carboplatin, and the culture was continued; (4) Second stage carboplatin induction: cells were cultured stably in medium containing 2 μM carboplatin for one week, and then the carboplatin concentration was gradually increased by 1 μM each time until the carboplatin concentration reached 10 μM; (5) Second stage shock: The concentration of carboplatin was instantly increased to 100 μM, and the cells were treated at this concentration for 24 hours. The culture medium was then replaced with a medium containing 20% ​​fetal bovine serum, and the cells were allowed to recover in a drug-free environment for 72 hours. The culture medium was then replaced with a medium containing 10 μM carboplatin and the cells were cultured continuously. (6) Phase III Carboplatin induction: After the cells have been cultured in a 10 μM carboplatin environment for one week, the concentration is increased by 2 μM each time until the carboplatin concentration reaches 20 μM, ensuring that the cells can grow and be passaged normally in the 20 μM carboplatin culture medium to obtain the target cell line; (7) Obtaining a stable cell line: The target cell line was repeatedly cultured in a 20 μM carboplatin environment, followed by two freezing and resuscitation treatments, and finally a stable carboplatin-resistant cell line was obtained.

4. The construction method according to claim 3, characterized in that: The specific operation of the freezing and reviving treatment in step (7) is: freezing the target cell line in liquid nitrogen for two weeks, then reviving and continuing to culture in a culture medium containing 20 μM carboplatin for one week.

5. Use of a breast cancer carboplatin-resistant cell line according to claim 1 in preparing a drug-resistant cell model.

6. Use of a breast cancer carboplatin-resistant cell line according to claim 1 in screening and preparing anti-breast cancer drugs.

7. Use of a breast cancer carboplatin-resistant cell line according to claim 1 in drug target development.

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